Tran et al. BMC Res Notes (2017) 10:148 DOI 10.1186/s13104-017-2471-6
RESEARCH ARTICLE
BMC Research Notes Open Access
Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte‑macrophage colony‑stimulating factor Anh‑Minh Tran1†, Thanh‑Thao Nguyen1†, Cong‑Thuan Nguyen1, Xuan‑Mai Huynh‑Thi1, Cao‑Tri Nguyen1, Minh‑Thuong Trinh1, Linh‑Thuoc Tran1, Stephanie P. Cartwright2, Roslyn M. Bill2 and Hieu Tran‑Van1*
Abstract Background: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemothera‑ pies. Recombinant hGM-CSF is produced industrially using the baker’s yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. Results: Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. Conclusion: Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production. Keywords: S. cerevisiae, P. pastoris, rhGM-CSF, Fermentation, Bioactivity, TF-1 cell Background Human granulocyte-macrophage colony-stimulating factor (hGM-CSF), a glycosylated cytokine, plays a vital role in proliferation and differentiation of granulocytes and macrophages from bone marrow progenitor cells [1]. *Correspondence: [email protected] † Anh-Minh Tran and Thanh-Thao Nguyen contributed equally to this work 1 Faculty of Biology and Biotechnology, University of Science, VNU-HCM, Ho Chi Minh, Vietnam Full list of author information is available at the end of the article
hGM-CSF also enhances cytotoxicity of macrophages and monocytes towards tumor cells [2], as well as tumor presentation of dendritic cells [1, 3]. Due to its stimulatory capacity on hematopoietic stem cells, recombinant hGM-CSF (rhGM-CSF) is recommended for therapeutic use in combination with chemo- or radio-therapy for cancer or transplantation patients [4]. hGM-CSF comprises 127 amino acids. The presence of 2 N- and 4 O-glycosylation sites leads to different glycoforms with molecular weights ranging from 14 to 60 kDa [3]. The glycosylation of the protein affects its
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Tran et al. BMC Res Notes (2017) 10:148
pharmacokinetics, in vivo half-life, immunogenicity and cytotoxicity [3]. rhGM-CSF is produced from bacteria, baker yeast or mammalian hosts [5]. rhGM-CSF produced in Escherichia coli is biologically active but unstable in human plasma. It induces an immune reaction because N-formyl methionine (fMet) is its first amino acid. rhGM-CSF produced in mammalian hosts has a similar glycosylation pattern to the native human protein, but production rates are slow [6, 7]. The product from baker’s yeast, Saccharomyces cerevisiae, is glycosylated and approved by the FDA for the treatment of neutropenia and leukemia in combination with chemo- or radio-therapy for cancer or transplantation patients. The drawbacks of using S. cerevisiae are hyper-glycosylated products and low cell density growth. The methylotrophic yeast Pichia pastoris has emerged as an alternative host due to its shorter and less immunogenic glycans, higher density cell growth and higher secreted protein yields than S. cerevisiae. In this study, we show that P. pastoris secretes a higher yield of more active recombinant rhGM-CSF than S. cerevisiae.
Methods In silico codon optimization
hGM-CSF coding sequences were from Gene Bank (Accession M11220). In silico codon optimization was done using DNA 2.0 software with P. pastoris and S. cerevisiae codon tables and optimized parameters [8]. The optimization evaluation was done via http://www.gcua. schoedl.de with low (
RESEARCH ARTICLE
BMC Research Notes Open Access
Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte‑macrophage colony‑stimulating factor Anh‑Minh Tran1†, Thanh‑Thao Nguyen1†, Cong‑Thuan Nguyen1, Xuan‑Mai Huynh‑Thi1, Cao‑Tri Nguyen1, Minh‑Thuong Trinh1, Linh‑Thuoc Tran1, Stephanie P. Cartwright2, Roslyn M. Bill2 and Hieu Tran‑Van1*
Abstract Background: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemothera‑ pies. Recombinant hGM-CSF is produced industrially using the baker’s yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. Results: Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. Conclusion: Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production. Keywords: S. cerevisiae, P. pastoris, rhGM-CSF, Fermentation, Bioactivity, TF-1 cell Background Human granulocyte-macrophage colony-stimulating factor (hGM-CSF), a glycosylated cytokine, plays a vital role in proliferation and differentiation of granulocytes and macrophages from bone marrow progenitor cells [1]. *Correspondence: [email protected] † Anh-Minh Tran and Thanh-Thao Nguyen contributed equally to this work 1 Faculty of Biology and Biotechnology, University of Science, VNU-HCM, Ho Chi Minh, Vietnam Full list of author information is available at the end of the article
hGM-CSF also enhances cytotoxicity of macrophages and monocytes towards tumor cells [2], as well as tumor presentation of dendritic cells [1, 3]. Due to its stimulatory capacity on hematopoietic stem cells, recombinant hGM-CSF (rhGM-CSF) is recommended for therapeutic use in combination with chemo- or radio-therapy for cancer or transplantation patients [4]. hGM-CSF comprises 127 amino acids. The presence of 2 N- and 4 O-glycosylation sites leads to different glycoforms with molecular weights ranging from 14 to 60 kDa [3]. The glycosylation of the protein affects its
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Tran et al. BMC Res Notes (2017) 10:148
pharmacokinetics, in vivo half-life, immunogenicity and cytotoxicity [3]. rhGM-CSF is produced from bacteria, baker yeast or mammalian hosts [5]. rhGM-CSF produced in Escherichia coli is biologically active but unstable in human plasma. It induces an immune reaction because N-formyl methionine (fMet) is its first amino acid. rhGM-CSF produced in mammalian hosts has a similar glycosylation pattern to the native human protein, but production rates are slow [6, 7]. The product from baker’s yeast, Saccharomyces cerevisiae, is glycosylated and approved by the FDA for the treatment of neutropenia and leukemia in combination with chemo- or radio-therapy for cancer or transplantation patients. The drawbacks of using S. cerevisiae are hyper-glycosylated products and low cell density growth. The methylotrophic yeast Pichia pastoris has emerged as an alternative host due to its shorter and less immunogenic glycans, higher density cell growth and higher secreted protein yields than S. cerevisiae. In this study, we show that P. pastoris secretes a higher yield of more active recombinant rhGM-CSF than S. cerevisiae.
Methods In silico codon optimization
hGM-CSF coding sequences were from Gene Bank (Accession M11220). In silico codon optimization was done using DNA 2.0 software with P. pastoris and S. cerevisiae codon tables and optimized parameters [8]. The optimization evaluation was done via http://www.gcua. schoedl.de with low (
